Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.

The gyrA and gyrB genes of Chlamydia pneumoniae TW-183 were cloned, and their proteins were purified by use of a fusion system with a maltose-binding protein. The 50% inhibitory concentrations of garenoxacin, sparfloxacin, moxifloxacin, gatifloxacin, and levofloxacin were 10.1, 47.5, 39.6, 64.2, and 156.9 g/ml, respectively, and the MICs against C. pneumoniae TW-183 were 0.008, 0.016, 0.063, 0.125, and 0.25 g/ml, respectively.Chlamydia pneumoniae is a frequent cause of communityacquired respiratory tract infections including pneumonia and bronchitis in adults and children (2,11,13). Quinolones have been recommended for the treatment of pneumonia caused by C. pneumoniae (1). The targets of quinolones are considered to be DNA gyrase and topoisomerase IV (7, 10), which are essential enzymes for controlling the topological state of DNA in DNA replication and transcription. DNA gyrase is composed of subunits A and B, encoded by the gyrA and gyrB genes, respectively. DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA.Quinolones developed recently, such as garenoxacin (T-3811, BMS-284756), moxifloxacin, and gatifloxacin, possess a wide antimicrobial spectrum (3,8,9,18) and also have a potent activity against C. pneumoniae (5, 6, 14, 15, 16, 17). These are expected to be useful in the treatment of infectious diseases caused by C. pneumoniae. In this study, we cloned C. pneumoniae DNA gyrase genes, and examined the activities of quinolones against the recombinant proteins to investigate the relation between the 50% inhibitory concentrations (IC 50 ) and the activities against C. pneumoniae.The gyrA and gyrB genes in C. pneumoniae CWL029 are denoted gyrA_1 and gyrA_2 and gyrB_1 and gyrB_2, respectively, in the GenBank (accession no.: AE001612 for gyrA_1 and gyrB_1 and AE001653 for gyrA_2 and gyrB_2). We first cloned the gyrA_1 and gyrB_1 genes from C. pneumoniae TW-183 and separately purified the encoded proteins (GyrA_1 and GyrB_1, respectively) by the use of a fusion system with a maltose-binding protein (MBP) and then examined enzymatic properties. For cloning gyrA_1 and gyrB_1 into the pT7Blue T vector, the C. pneumoniae TW-183 genome was isolated from chlamydial elementary bodies (10 4 inclusion-forming units). PCR amplification of gyrA_1 and gyrB_1 was performed with the CPGA-1-CPGA-2 and CPGB-1-CPGB-2 primer sets, respectively (Table 1). DNA was amplified for 35 cycles, in which the conditions were 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. Amplified PCR products for CPGA-1-CPGA-2 and CPGB-1-CPGB-2 primer pairs were about 2.5 and 2.4 kbp, respectively, and cloned genes were sequenced. Two sets of oligonucleotide primers (CPGA-3-CPGA-4 and CPGB-3-CPGB-4; Table 1) were designed for amplification of gyrA_1 and gyrB_1 genes and their subsequent insertion into BamHI and HindIII sites and BamHI and PstI sites, respectively, in the pMAL-c2 vector. The proteins obtained by fusion of MBP with GyrA_1 and GyrB_1 (GyrA_1-MBP and GyrB_1-MBP, respectively) were produced by the Escherichia coli DH5␣ overexpres...

The envelope of rabies virus (a prototype member of the Rhabdovirus family) contains two kinds of major viral proteins, a transmembrane glycoprotein (G) and a matrix protein (M; formerly called M2), as well as some other host-derived minor components such as CD44 and CD99-related glycoprotein (VAP21) (11,(23)(24)(25). The M protein is a small-sized nonglycosylated internal protein (24-kDa), and is thought to be located underneath the lipid bilayer of the viral envelope where the protein is associated in some way with both the viral G protein and ribonucleoprotein (RNP) (26). Although the M protein is one of the major viral envelope components, detailed studies on the M protein have not been so frequently

The envelope of the rabies virus (a prototype of the Rhabdoviridae) contains two species of virus-coded major polypeptides; a transmembrane glycoprotein (G) and a matrix protein (M; formerly called M2), and some host-derived minor components including CD44 and a CD99-related glycoprotein (VAP21) (20,36,37,40). The M protein is a small non-glycosylated internal virion protein (Mr.ϭ24 kDa), and is thought to be located underneath the lipid bilayer of the envelope, where the M protein is thought to associate with the viral G protein and also the viral nucleoprotein (RNP) (41).The G protein has been studied extensively for many years because of its possible roles in determining the host range, neurovirulence, and immunogenicity of the virus as well as important roles in the virus replication cycle